The scientific art of plant breeding is aimed at the development of plants with new or modified traits. Plant breeding involves two main phases--the creation of genotypic variability and the selection of plants with particularly desired genotypes. Today, the penumbra of plant breeding encompasses both traditional methods of producing genotypic variability (crossing two plant lines to introduce traits from one to the other) and newer methods of genetic engineering. It is to such genetic engineering techniques that the present invention is directed.
Genetic engineering provides plant breeders with new tools with which to manipulate plant genomes. Rather than move large segments of plant genomes from one line to another by crossing related plant species, plant breeders can now introduce individually characterized genes into a particular plant line by genetic engineering. This technique not only provides a level of specificity not previously available, it also facilitates the transfer of genes into plants from completely unrelated plant species and indeed from viruses, bacteria, and animals.
Genetic engineering offers the hope of modifying many plant characteristics, including crop yield, stress tolerance and pathogen resistance. The regulation of gene expression is a key aspect of genetic engineering, and it is to this issue that the present invention is directed.
Plant viruses are responsible for major losses in worldwide crop production. Much effort is directed towards the development of new plant varieties which exhibit increased resistance to viral infection. Until recently such efforts were primarily based on the traditional plant breeding approach, however this approach is often limited by a lack of sources of resistance within the crop species. The advent of modern molecular biology techniques has facilitated the development of new methods of rendering plant varieties resistant to virus attack that are not limited by a requirement for preexisting resistance genes within a species.
Molecular Approaches
Many of these molecular approaches are based on the theory of pathogen derived resistance (Sanford and Johnston, 1985). This theory predicts that a "normal" host (plant)--pathogen (virus) relationship can be disrupted if the host organism expresses essential pathogen derived genes. It has been proposed that host organisms expressing pathogen gene products in excess amounts, at an inappropriate developmental stage, or in a dysfunctional form may disrupt the normal replicative cycle of the pathogen and result in an attenuated or aborted infection of the host.
Two approaches typify this pathogen derived resistance: coat protein mediated resistance and antisense RNA expression. Coat protein mediated resistance involves the production of transgenic plants expressing the coat protein gene of a particular virus. These plants may show an increased resistance to infection by that virus type. Coat protein mediated resistance has been demonstrated for several virus groups. While the mechanism of this resistance is not yet fully understood, it has been suggested that the presence of the plant synthesized coat protein prevents the removal of the protein coat (uncoating) of an invading virus and/or virus movement within the infected plant, leading to resistance. A major concern with this approach is the possible interaction between the viral coat protein expressed in the plant and other viral nucleic acids, which could potentially produce new strains of virus.
Antisense RNA technology involves the production of an RNA molecule that is complementary to the messenger RNA molecule of a target gene; the antisense RNA can potentially block all expression of the targeted gene. In the anti-virus context, plants are made to express an antisense RNA molecule corresponding to a viral RNA (that is, the antisense RNA is an RNA molecule which is complementary to a plus sense RNA species encoded by an infecting virus). Such plants may show a slightly decreased susceptibility to infection by that virus. Such a complementary RNA molecule is termed antisense RNA. It is thought that the plant encoded antisense RNA binds to the viral RNA and thus inhibits its function. This approach has only met with very limited success.
Potyviruses
The Potato Virus Y, or potyvirus, family represents a large number of plant viral pathogens which collectively can infect most crop species including both monocotyledonous and dicotyledonous plants. Potyvirus infection can induce a variety of symptoms including leaf mottling, seed and fruit distortion and can severely compromise crop yield and/or quality (Hollings and Brunt, 1981).
Potyviruses comprise a large family of aphid transmitted plant viruses that are members of the picornavirus superfamily (Goldbach, 1987). In general, potyviruses are flexous rod-shaped viruses with a single-strand plus sense RNA genome of circa 10,000 nucleotides which has a viral encoded protein linked to the 5' end and a 3' polyadenylate region. A single open reading frame codes for a polyprotein of approximately 350 kDa which is proteolytically processed into mature viral gene products. Nine individual gene products are released from the polyprotein by three proteinases which are part of the polyprotein (Riechmann et al., 1992).
The potyvirus RNA genome is encapsidated by approximately 2,000 copies of a coat protein monomer to form a virion. This capsid protein is encoded by the sequence present at the 3' end of the large open reading frame.
Potyviruses can be transmitted by aphids and in some instances can also be transmitted in the seeds of infected plants. Replication of the viral RNA is thought to occur in the cytoplasm of infected plant cells after uncoating. The replication mechanism involves both translation of the plus sense RNA to yield viral gene products (which include a replicase and a proteinase) and also the synthesis of a minus sense RNA strand. This minus sense strand then acts as a template for the synthesis of many plus sense genomes which are subsequently encapsidated in coat protein to yield infectious mature "virions," thus completing the replicative cycle of the virus.
Experiments have been reported in which transgenic plants expressing the coat protein gene of a potyvirus show a reduced susceptibility to virus infection (Lawson et al. 1990; Ling et al. 1991; Stark and Beachy 1989).
It is an object of the present invention to provide new methods of regulating gene expression that can be used to produce plants with a reduced susceptibility to virus infection.